The intracellular bacillus pathogen Mycobacterium tuberculosis (M. tb) causes the fatal and rapidly spreading disease tuberculosis (TB) in non-human primates (NHP), similar to active TB in humans. In NHPs, M. tb is typically spread by inhalation of aerosolized infectious particles expelled by infected individuals (humans and animals) [1]. Because of the health threat to captive primates and the zoonotic potential to and from their human caretakers, strict screening protocols have been implemented to prevent outbreaks[2]. Despite the precautions taken to prevent M. tb exposure in captive primate colonies, outbreaks continue to cause a major impact on colony health. Outbreaks result in animal losses, disruption of research, potential exposure to human caretakers, and the immeasurable costs to contain the outbreaks [3-11].
Current methods for TB screening in non-human primates rely on the bi-annual intradermal tuberculin skin test (TST), a test that has been routinely used for decades [2, 12]. Injection of M. tb antigen is typically performed in the eyelid, and relies on immune cell mediated recognition of the antigens generated by previous exposure to M. tb. The antigens currently used in the TST are semi-purified heterogeneous crude protein preparations of either mammalian old tuberculin (MOT) or purified protein derivative (PPD). The cell mediated response resulting in local inflammation and necrotic tissue measured by a TST takes at least 4 weeks after infection to develop and has some severe limitations in terms of diagnostic sensitivity and specificity. Additionally, the TST is labor intensive, and requires increased animal handling over the three days it takes to develop the response of a swelled eyelid or necrotic tissue.
While false positive TST results in loss of individually valuable research animals, the greatest threat to NHP colony and human health are false negative results, which make entire colonies vulnerable to outbreaks [5, 10, 13]. Because NHPs can develop latent TB without obvious clinical symptoms [3, 7, 14-15], false negative results or anergy to TST pose a significant threat to colony health when combined with the low diagnostic sensitivity of the TST. Anergy can also be induced in research primates by immunosuppressive treatment or disease, or concurrent viral infection like measles [4, 9, 16-17]. Additionally, reports of anergy to TST following measles vaccination complicates using the TST as part of importation quarantine procedure[18]. Despite the TST being USDA-approved for diagnostic testing of NHPs for TB, it is widely considered that a negative TST result does not reliably exclude tuberculosis infection (reports between 79 and 90% predictive value)[19]. It is, however, the standard for antemortem diagnostic testing and requires lengthy postmortem confirmation by the “gold standard” of mycobacterial culture and isolation.
Other immune-based screening assays and serological tests, including interferon gamma release assays (IGRA) and commercial antibody detection assays can have a higher sensitivity than the TST for acute infection, but rely on similar sets of M. tb recombinant proteins that often fail to identify latently infected animals. In the case of the commercially available IGRA test PRIMAGAM (Prionics AG, Zurich, Switzerland), the semi-purified PPD antigen set is used to stimulate IFN-γ release by whole blood leukocytes[19]. The PrimaTB STAT-PAK assay is a lateral flow device for measuring presence of TB-specific antibodies in NHP plasma or serum, and uses an antigen cocktail of recombinant antigens including ESAT-6, CFP-10, MPB83, and TBF10 (a fusion protein from Corixa Corp)[20]. Relative to the TST these tests are expensive, technically difficult, and impractical to perform on a colony-wide basis[19]. Moreover, these methods all rely on semi-purified or recombinant antigens for identifying a biological or antibody response, all are subject to the same limitations of sensitivity. At best, reported predictive value in experimentally infected animals is between 83 and 94% for PRIMAGAM and 80 and 94% for PrimaTB STAT-PAK, respectively [19-21]. Unpublished studies have reported significantly lower predictive value (<50%) for naturally occurring infections in NHPs (Table 2). Current recommendations for TB testing include routine TST in conjunction with IGRA or antibody assays for routine screening because no one test is both sensitive and specific enough[22].
What is needed in the art are improved devices and methods for determining if a subject animal has tuberculosis, and especially devices and methods which allow for accurate, specific and sensitive monitoring of primate colonies.